Inducible nitric oxide synthase (NOS2) and cyclooxygenase-2 are involved in wound healing, angiogenesis, and carcinogenesis. NOS2 up-regulation and increased nitric oxide production also affects the redox state of cells and induces protein, lipid, and DNA modifications. Recent research by our laboratory led to the novel and clinically significant observation that NOS2 expression is associated with a prognostic basal-like transcription pattern and is an independent predictor of poor survival in women with estrogen receptor-negative breast tumors. NOS2 remained a predictor of poor survival when the analysis was restricted to only those patients with basal-like breast tumors. This result suggests that in addition to inducing a basal-like signature in estrogen receptor-negative tumors, NOS2 may further enhance disease aggressiveness in the presence of this signature. Increased NOS2 in breast tumors also correlated with other poor outcome markers, such as an increased tumor vascularization and p53 mutation frequency, and activated EGFR. Both p53 mutations and EGFR overexpression occur more commonly in basal-like breast tumors than other breast cancer subtypes. Additional work showed that nitric oxide activates epidermal growth factor receptor, consistent with previous findings in lung cancer, and induces proteins, such as CD44 and c-Myc, in estrogen receptor-negative human breast cancer cells. These markers have been linked to an embryonic stem cell-like phenotype in breast cancer and disease outcome. Recently, an increased expression of stem cell markers in basal-like tumors has been reported. Among them was CD44, which is a receptor for hyaluronan and osteopontin. CD44 is a poor outcome marker in breast cancer and CD44-positive breast cancer cells have an increased invasive activity, increased resistance to radiation therapy and chemotherapeutics. Together, these novel observations link NOS2 to the development of a poorly differentiated breast cancer phenotype with stem cell-like characteristics. Nitric oxide may induce this phenotype by activation of c-Myc or by inducing the release of stem cell renewal factors like IL-8, a property that nitric oxide has, as we have shown. We propose that NOS2 and downstream targets of NOS2 signaling are novel therapeutic targets for estrogen receptor-negative breast cancer in general, and more specifically for basal-like breast cancer and the triple-negative disease. We also completed a study re-examining cyclooxygenase-2 (COX2) as a marker of poor outcome in breast cancer. It has previously been reported that COX2 is a marker of outcome and a therapeutic target in the estrogen receptor-positive disease but very recent clinical trials failed to find a clear benefit of COX2 inhibitors for patients with estrogen receptor-positive breast cancer. In our study of 248 women with incident breast cancer from the Greater Baltimore area, increased expression of COX-2 was associated with decreased breast cancer-specific survival only in patients with estrogen receptor-negative and HER2-positive tumors, respectively. In patients with both estrogen receptor-negative and HER2-positive tumors, increased tumor COX-2 was associated with the most inferior survival among all patient groups, as judged by the hazard ratio in the multivariate analysis. This was accompanied by increased Akt pathway activation, as judged by the phosphorylation status of Akt and two key downstream targets in the apoptosis pathway. These findings could have implications for COX-2 targeted therapy in breast cancer and suggest that patients with estrogen receptor-negative and HER2-positive tumors would benefit from a COX-2 targeted therapy with the efficacy of this therapy being strongest in patients with both an estrogen receptor-negative tumor status and an amplification of HER2 leading to high HER2 expression. In FY11, we continued to comprehensively examine the metabolome, proteome and transcriptome of estrogen receptor-positive and estrogen receptor-negative breast tumors from African-American and European-American patients for biomarker discovery. This project received funds through a NCI Director Innovation Award. The promise of the study is the discovery of novel biomarkers for prognosis, and for elucidating what may drive the aggressiveness of breast cancer in African-American women. To date, we have completed the metabolome and transcriptome analysis of the tumors and the adjacent non-cancerous tissues and have collected the raw data from the proteome analysis for about 75% of the tissues. The transcriptome data are being analyzed and compared with data from a previously published pilot study (Martin et al. PLoS One, 2009), showing significant gene expression differences between tumors from African-American and European-American breast cancer patients. The analysis of the metabolome yielded preliminary information on 536 biochemicals in the breast tumors and the surrounding normal tissues. Of those, 333 are named/identified. 360 biochemicals differed significantly in their concentrations between estrogen receptor-negative and normal surrounding tissue. 311 differed in their concentrations between estrogen receptor-positive and normal surrounding tissue. There were significant differences in metabolite levels between breast cancer subtypes and tumors from African-American patients and European-American patients, specifically in estrogen receptor-negative tumors. We did not find any differences in tissue metabolite concentrations by body mass index, menopausal status, or by tumor stage and tumor HER2 status. There are perhaps also differences by income and education of the patients, a finding which will need further examination. Currently, we are validating these findings in a follow-up study consisting of 106 estrogen receptor-negative tumors and adjacent non-cancerous tissues (as available) from African-American (n = 49) and European-American breast cancer patients (n = 21). In this study, we will evaluate the abundance of those 80-150 metabolites that showed the most significant differences in the pilot study between tumor and normal and between African-American and European-American patients. This validation study is conducted at the Baylor College of Medicine in collaboration with Arun Sreekumar, using targeted assays. In addition, we are examining the influence of candidate metabolites on cancer phenotypes in cell culture systems. Lastly, we started a new project evaluating the impact of stressful life events on tumor biology. In cell culture systems, we will model the effects of beta adrenergic signaling on tumor stroma interactions. In a clinical study, we will give breast cancer patients, who have breast cancer surgery, a short survey evaluating their perceived stress within the last one month. We will also collect frozen tumor and adjacent normal breast tissue and blood samples from these patients and evaluate whether the breast tissue or the blood samples have a biological signatures related to their perceived stress. We hypothesize that patients with a high perceived stress exposure have a biological signature consistent with a more aggressive disease and poorer survival. The pilot study is designed to collect 100 tumor/normal pairs from consented patients with a completed survey.